Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 surface expression and number of antigens per cell (APC) on primary AML bone marrow samples. Data shown for AML blasts (CD45 dim SSC lo ; n=25) and leukemic stem cell (LSC)-enriched compartments (CD34 + CD38 - ; n=20). Each data point represents an individual patient. Quantification of ADGRE2 expression was performed using a standard curve generated by QuantiBRITE beads and interpolation of geometric mean fluorescence intensity values of positively expressing cells. Data represented as box-and-whisker plots indicating the 25 th , 50 th (median), and 75 th percentiles; whiskers denote minimum and maximum values. (B) EMR2-directed binders were identified by phage display panning against single-chain variable fragment (scFv) and heavy chain variable region (VH) libraries. Binder kinetic and affinity characterization (EC50 and KD) were performed by multipoint flow cytometric, ELISA and Blitz analyses. (C) Schematic representation of ADGRE2 full-length (FL) and splice isoforms, FL CD97, and a chimeric construct in which the native ADGRE2 GAIN and GPS domains were replaced with the corresponding regions from CD97. Illustration created using BioRender. (D) Binding specificity of ADGRE2-directed CAR binders to ADGRE2 isoforms, FL CD97, and the chimeric ADGRE2-CD97 construct (GAIN and GPS replacement) expressed on the surface of HEK293T cells following transient transfection. CAR constructs were tagged with a C-terminal FLAG peptide; binding was assessed by flow cytometry and anti-FLAG secondary detection. (E-F) In vitro cytotoxicity of ADGRE2-directed CAR Ts (ADGRE2-1-scFv, ADGRE2-2-scFv, ADGRE2-5-scFv, ADGRE2-6-VH, ADGRE2-8-VH, or ADGRE2-9-VH) or untransduced T cells (UTD) against MOLM-13 wild-type (WT) and ADGRE2-knockout (KO) targets at an effector-to-target (E:T) ratio of 1:1. Cytotoxicity assessed at 24h and 48h by flow cytometry; viable targets defined as Annexin-V - /LIVE-DEAD - . Each data point represents an individual T cell donor; data shown as mean ± SD from 2-3 donors. Two-way ANOVA with Šidák multiple comparisons test was performed to compare viability of targets in co-culture with CAR T cells to UTD, averaging both timepoints; *p < 0.05, **<0.01, ***p < 0.001, ****p<0.0001. (G) T cell activation measured by CD25 surface expression after 48h co-culture. UTD and effector-alone conditions served as controls. Data shown as mean ± SD from 2-3 donors; each data point represents a donor. (H) Cytokine secretion profile of ADGRE2-directed CAR Ts and UTD controls following 48 h co-culture with MOLM-13 WT or ADGRE2-KO targets at a 1:1 E:T ratio. Supernatants were analyzed for IFNγ, TNFα, IL-2, sFasL, and Granzyme B. Effector-alone controls included. Data shown as mean ± SD from 1-3 donors, each dot represents a donor. Abbreviations: scFv, single-chain variable fragment; VH, heavy chain variable domain; GAIN, G-protein-coupled receptor (GPCR) autoproteolysis-inducing domain; GPS, GPCR proteolytic site; 7TM, 7-pass transmembrane domain; SD, standard deviation; ns, not significant; IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; IL-2, interleukin-2; sFasL, soluble Fas ligand; N.D., not detected.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Generated, Fluorescence, Whisker Assay, Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Transfection, Flow Cytometry, In Vitro, Knock-Out, Co-Culture Assay, Activation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 surface expression on MOLM-13 wild-type (WT) and lentivirus engineered clones. ADGRE2 surface quantification was completed using QuantiBRITE bead interpolation. Data shown as mean ± SD from n=3 independent measurements. (B) Representative flow cytometry plots of ADGRE2-5-scFv CAR surface expression (anti-human IgG H+L) from n=3 T cell donors. (C) Geometric mean fluorescence intensity (gMFI) of ADGRE2-5-scFv CAR expression in transduced T cells from panel (B). Data shown as mean ± SD; each data point represents a technical replicate (n=3). (D) In vitro cytotoxicity assessment of untransduced (UTD) or ADGRE2-5-scFv (CAR) T cells against MOLM-13 ADGRE2-KO, Clone 1, Clone 2, or WT targets at a 1:4 effector-to-target (E:T) ratio. Viability was assessed after 48 h by flow cytometry (Annexin-V - /LIVE-DEAD - ). Target-alone cultures served as controls. Data represented as mean ± SD from n=3 T cell donors. (E) ADGRE2-specific killing of MOLM-13 clones and WT at a 1:4 E:T ratio, calculated as ( Viability untreated – Viability treated )/ Viability untreated x 100 followed by subtracting killing observed in KO targets for background correction. Data represented as mean ± SD from n=3 T cell donors. (F) Effector activation measured by CD25 surface expression after 48 h co-culture with indicated MOLM-13 targets; flow cytometry analysis. Effector-alone condition included as a control. Data shown as mean ± SD from n=3 donors. (G) Correlation between ADGRE2-5-scFv T cell activation (CD25 + ) and ADGRE2 surface intensity (antigens per cell, APC) on MOLM-13 targets at a 1:4 effector-to-target (E:T) ratio. Data shown as mean ± SD. Pearson correlation efficient (r) and linear regression analysis were generated using GraphPad Prism. Abbreviations: SD, standard deviation; KO, knockout.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Clone Assay, Flow Cytometry, Fluorescence, In Vitro, Activation Assay, Co-Culture Assay, Control, Generated, Standard Deviation, Knock-Out

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 percent surface expression and antigens per cell (APC) on healthy bone marrow (n=3) and peripheral blood (n=5) hematopoietic cell subsets. Quantification of ADGRE2 expression was performed by interpolation of PE geometric mean fluorescence intensity (gMFI) values using a QuantiBRITE bead-generated standard curve. Data represented as median values; each point represents an individual donor sample. One donor showed undetectable ADGRE2 expression on lymphocytes by QuantiBRITE interpolation. (B) Heatmap summarizing median ADGRE2 APC across healthy hematopoietic and AML disease populations. (C) Schematic of ADGRE2 gene structure (not drawn to scale) with indicated start/stop codons (triangles) and associated coding regions. Positions of reported homozygous predicted loss-of-function (pLOF) variants and the number of affected individuals are indicated (arrows). Exonic regions analyzed by ddPCR are also shown. (D) Summary table of ADGRE2 homozygous pLOF natural variants identified in gnomAD v4.1.0. (E) Schematic of full-length ADGRE2 protein with indicated locations of pLOF variants (A-H; arrows). Wild-type (WT) and variant coding sequences were cloned into pcDNA3.1(+)-IRES-eGFP expression plasmids, with an N-terminal HA tag inserted in each construct. General binding regions for the anti-ADGRE2 (clone 2A1) and anti-HA antibody (clone 16B12) used in flow cytometry and Western blotting are shown. (F) Surface expression (geometric mean fluorescence intensity, gMFI) of ADGRE2 and HA in HEK293T cells transfected with WT and variant (A-H) ADGRE2 plasmids; measured by flow cytometry 24 h post-transfection and normalized to WT. Empty vector (em.) and non-transfected cells served as controls. Data represented as mean ± SD; n=2 biological replicates. Representative Western blot of total ADGRE2 and HA protein expression; β-actin served as a loading control. (G) Transcript expression of ADGRE2 exons 10-12 and exon 6 in healthy bone marrow (BM; n=6), CD34 + hematopoietic stem and progenitor cells (HSPC) day 2 post-thaw (n=9), and AML bone marrow samples at diagnosis (n=10) and relapse (n=10); measured by digital droplet PCR (ddPCR) and normalized to GUSB (glucuronidase beta) for each sample. (H) Base editing efficiency of Variants D and H in CD34 + HSPCs using BE4-PpAPOBEC1 or BE4-PpAPOBEC-SpG guide-RNAs targeting respective variant regions. Editing was assessed on day 5 post-electroporation by next-generation-sequencing (rhAmpSeq). “On-Target” represents reads with the intended C-to-T edit and no bystander conversions; “Bystander” includes reads with off-target C-to-T conversions alone or in combination with the intended on-target edit. ADGRE2 surface expression was assessed in parallel by flow cytometry and normalized to non-edited control HSPC cells; n=1 biological replicate. Abbreviations: HSC, hematopoietic stem cells; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythroid progenitor; CLP, common lymphoid progenitor; cDCs, classical dendritic cell; pDCs, plasmacytoid dendritic cells; NK, natural killer cell; HSPC, hematopoietic stem and progenitor cells; ddPCR, digital droplet PCR; SD, standard deviation.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Fluorescence, Generated, Variant Assay, Clone Assay, Construct, Binding Assay, Flow Cytometry, Western Blot, Transfection, Plasmid Preparation, Control, Biomarker Discovery, Electroporation, Next-Generation Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) CD34 + hematopoietic stem and progenitor cells (HSPCs) were electroporated with ADGRE2-targeting CRISPR-Cas9-ribonucleoprotein (RNP) complex (KO-Cas9) or a non-editing control RNP (Control). Two days post-electroporation (EP), cells were cultured for 14 days under monocytic differentiation conditions (indicated by dashed line and shaded grey). Editing frequency was assessed by ICE analysis and ADGRE2 surface expression was measured by flow cytometry. Data shown as mean ± SD; n=2 donors. (B) Cell viability and concentration throughout the 16-day in vitro culture. Quantified by cell counter; data shown as mean ± SD; n=2 donors. (C) Phenotypic analysis of differentiating cells by flow cytometry using CD11b (pan-myeloid marker), CD33 (myeloid marker), CD14 (monocytic lineage marker), and CD15 (granulocytic lineage marker). Data shown as mean ± SD; n=2 donors. (D) Inflammatory cytokine secretion (IL-6, MIP-1α, TNFα) evaluated in monocytic differentiated cells, collected from supernatant at study end. Data shown as mean ± SD; each dot represents a donor. Statistical analysis was performed using two-way ANOVA comparing basal condition versus LPS stimulation, or basal condition versus R848 (TLR7/8 agonist) stimulation; *p < 0.05, ***p < 0.001. Two-way ANOVA with Šidák multiple comparisons test was performed to compare KO-Cas9 to Control for each stimulation condition. (E) Schematic representation of the lead ABE8.20-m guide-RNA (gRNA) targeting the splice donor site at the end of exon 19 of ADGRE2 . On-target A-to-G editing site is bolded and indicated by an arrow. (F) CD34 + HSPC were electroporated with ABE8.20-m mRNA and either an ADGRE2 -targeting gRNA (KO-ABE) or non-targeting control (Control) and cultured for 5 days. Editing outcomes—splice-site disruption (intended A-to-G edit), missense conversions, and insertions/deletions (InDels)—were quantified by next-generation sequencing (rhAmpSeq). ADGRE2 surface protein expression was measured by flow cytometry. Data represented as mean ± SD; n=4 donors. (G) Frequency of HSPC subpopulations 48 h post-electroporation (KO-ABE and Control), measured by flow cytometry. Subsets included CMPs, MPPs, MLPs, and LT-HSCs, expressed as percentage of total live cells. Cells were bulk sorted and editing frequency of splice-site disruption was assessed by rhAmpSeq. Data shown as mean ± SD; n=2 donors. Statistical analysis by ANOVA with Šidák multiple comparisons test. Abbreviations: KO, knockout; SD, standard deviation; IL-6, interleukin-6; MIP-1α, macrophage inflammatory protein-1 alpha; TNFα, tumor necrosis factor alpha; LPS, lipopolysaccharide; TLR, toll-like receptor; CMP, common myeloid progenitors; MPP, multipotent progenitors; MLP, multi-lymphoid progenitors; LT-HSCs, long-term hematopoietic stem cells.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: CRISPR, Control, Electroporation, Cell Culture, Expressing, Flow Cytometry, Concentration Assay, In Vitro, Marker, Disruption, Next-Generation Sequencing, Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Experimental schema for 16-week xenotransplantation study in NSG mice. (B) KO-ABE and Control (CTR) HSPCs were frozen 48 h post-electroporation and then thawed for same-day injection into NSG mice (n=10 mice per group). Bone marrow (BM) was harvested after 16 weeks. On-target editing (A-to-G conversion at desired splice donor site) was assessed in input cells (pre-freeze) and BM samples by next-generation sequencing (rhAmpSeq). Black square indicates input material; each purple dot represents an individual mouse BM sample and horizontal bar denotes group mean. (C) Chimerism and multilineage reconstitution in BM, 16 weeks post-engraftment; measured by flow cytometry. Human chimerism calculated as hCD45 + / (hCD45 + + mCD45 + ) x 100. Frequencies of CD34 + HSPC, myeloid lineages (Monocytes, pDCs, Neutrophils, cDCs, Basophils, Mast Cells), and lymphoid (T cells, CD3 + ; B-cell, CD19 + ) populations were measured within hCD45 + compartment. CD97 + cells also assessed to confirm editing specificity. Data shown mean ± SD; n=10 mice per group. Each dot represents an individual mouse BM sample. Statistical analysis by two-way ANOVA; ns = not significant (p > 0.05). (D) ADGRE2 surface protein expression in total bone marrow hCD45 + , myeloid, lymphoid, and CD97 + populations in KO-ABE versus CTR mice at study end; measured by flow cytometry. Data represented as mean ± SD; n=10 mice per arm, each dot represents one mouse. Statistical analysis performed using two-way ANOVA; ****p<0.0001. Abbreviations: NSG, NOD-scid IL2Rg null; KO, knockout; HSPC, hematopoietic stem and progenitor cells; hCD45, human CD45; mCD45, murine CD45; pDCs, plasmacytoid dendritic cells; cDCs, classical dendritic cells; SD, standard deviation.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Control, Electroporation, Injection, Next-Generation Sequencing, Flow Cytometry, Expressing, Knock-Out, Standard Deviation